N-linked glycosylation is the major non-protein modification that occurs on membrane and secretory glycoproteins and therefore involves a major and important series of reactions. In spite of the widespread distribution of this pathway, very little is known about the regulation of these reactions, nor on how the entire pathway is controlled. In addition, very few of the enzymes involved in the pathway have been purified or characterized. The PI has evidence that the pyrophosphorylases that synthesize GDP-mannose (GDPMPP) and UDP-G1cNAc (UDPHexNAccPP), two key precursors of N- and O-glycosylation, have unusual substrate specificities and other properties that suggest that they play a regulatory role in carbohydrate metabolism and/or biosynthesis. In addition, the UDPHexNAcPP, which is membrane-bound, may reside in a complex linked to other ER or Golgi proteins and this complex may regulate G1cNAc transfer. In this study, the PI will purify GDPMPP and determine whether one subunit synthesizes GDP-glucose and the other GDP-mannose, and what regulates these activities and how. The PI will clone and express GDPMPP and then delete or alter the activity to determine its role in N-glycosylation. Similar experiments will be done with membrane-associated UDPHexNAcPP. The PI will use these pyrophosphorylases to prepare photoprobes, such as N3-UDP [32P]-G1cNAc, to isolate the transferases involved in N-linked glycosylation so that their properties and roles in regulation of glycosylation can be assessed. These enzymes are likely to be important in metabolic disease such as diabetes, atherosclerosis and glycoprotein deficiency syndromes.